Vectors and transformed host cells for recombinant protein production at reduced temperatures

ABSTRACT

The invention relates to an expression vector, the nucleic acid sequence of which comprises a promoter that is capable of controlling, when the vector is in a bacterial host and the temperature is lowered to below about 20° C., the production of recombinant peptide or protein encoded by a gene contained within the vector. The invention also relates to host cells containing the vectors and to a method of producing a recombinant protein.

This application is a continuation in part of U.S. Ser. No. 08/278,281,filed on Jul. 21, 1994, now U.S. Pat. No. 5,654,169.

TECHNICAL FIELD OF THE INVENTION

The invention relates to an expression vector, the nucleic acid sequenceof which comprises a promoter that is capable of controlling, when thevector is in a bacterial host and the temperature is lowered to belowabout 20° C., the production of recombinant peptide or protein encodedby a gene contained within the vector. The invention also relates totransformed host cells containing the vectors, and to a method ofproducing a recombinant protein.

BACKGROUND OF THE INVENTION

Genetic engineering provides methods for the cloning of foreign genes,their cDNA sequences or portions thereof, and their introduction intobacterial host cells such as Escherichia coli. The level of productionof a foreign protein in the host cell depends on appropriately arrangedtranscription and translation control sequences that permit theregulated expression of the desired proteins or peptides coded by theforeign genes, gene fragments, various gene fusions or mutants thereof.

Transcription (i.e., the process leading to the production of mRNA) is akey step in the expression of a gene. Transcription initiation occurs atspecific promoters which regulate gene expression. Promoters areregulated negatively by repressors and positively by activator proteins.The initiation of transcription can be separated into several steps (Bucet al., Biochemistry (1985) 24:2712). In order to act effectively,promoters are made of three elements. A core sequence is recognized byRNA polymerase. This region is flanked by the USR, or the upstreamregion, and the DSR, or the downstream region. The USR was shown to bindspecific activator proteins (Adhya et al., Gene (1993) 132:1). Two modesof gene activation are generally considered. DNA-bound activator makesdirect contact with RNA polymerase and thereby facilitates the bindingand isomerization of RNA polymerase. Alternately, the bound activatorchanges the structure of the promoter, thereby favoring transcriptioninitiation.

In order to maintain the desired foreign gene and to express it in abacterial host cell at a high level for protein purification, it isessential to place the gene under the control of a regulated promoterthat permits turning off of gene expression during the first stage offermentation in which the cell mass is increased, and turning it on atthe second fermentation stage in which maximal gene expression andprotein production is needed.

Production of proteins utilizing a number of promoters and ribosomebinding sites has been previously described. For example, the use of thepL promoter for regulated gene expression has been the subject ofseveral references (e.g., Bernard et al., Gene (1970) 5:59; Derom etal., Gene (1982) 17:45; Gheysen et al., Gene (1982) 17:55; Hedgpeth etal., Mol. Gen. Genet. (1978) 163:197; Remaut et al., Gene (1981) 15:81;and Deryneck et al., Nature (1980) 287). A number of ribosome bindingsites have been employed in order to obtain a high level of proteinsynthesis, such as the phage λ cII translation initiation region usedfor the production of cII (Oppenheim et al., J. Mol. Biol. (1982)158:327; Shimatake et al., Nature (1981) 292:128). Similarly, productionof human growth hormone (hGH) and bovine growth hormone (bGH) aredescribed, inter alia, in U.S. Pat. No. 4,997,916.

One of the major problems in producing eukaryotic proteins in thebacterium E. coli is that the desired protein is often obtained ininactive aggregates termed inclusion bodies. The inclusion bodies canfacilitate protein purification provided that it is possible to refoldthe desired peptide to the active protein form in vitro by some process(see, for example, U.S. Pat. No. 4,997,916). However, there are proteinsfor which in vitro refolding is highly inefficient.

Several factors appear to contribute to the formation of inclusionbodies. The most important of these are the rapid synthesis of a singleprotein following induction of gene expression in the fermentationprocess, and the temperature at which fermentation takes place. At hightemperatures, refolding of the individual molecule is slowed down. Thepresence of a high concentration of incompletely folded proteins leadsto protein aggregation due to the intermolecular interactions ofhydrophobic domains. In production of the native protein, thesehydrophobic domains play an important role in the folding of the proteininto a fully active form.

At present, there is no known way to direct the production of proteinsin their native form during the fermentation process. For some proteins(such as hGH and bGH) it is possible to disaggregate and refold thedesired protein, while for others there is no known refolding process.However, it is known that chaperonin protein complexes can act tofacilitate disaggregation in vivo. This process can also be carried outon a small scale in biochemical laboratories.

It has been shown that proteins expressed in E. coli at low temperatureshave an increased solubility (Schein et al., Bio/Technology (1988)6:291; Schein, Bio/Technology (1989) 7:1141). For example, wheninterferon (IFN-α2) was expressed in a bacterial culture at 37° C., 95%of the protein was found in inclusion bodies. In contrast, only 27% ofthe protein was insoluble when the same culture was grown at 30° C.Similar results were obtained for IfN-γ, and Shirano et al. (FEBS (1990)1:128) demonstrated the low-level expression of soluble lipoxygenease inE. coli at about 15° C.

Accordingly, there remains a need for vectors which allow for theisolation of biologically active eukaryotic protein from bacterialcells. It is an object of the present invention to provide such vectors,as well as methods for the production and isolation of eukaryoticproteins from bacterial cells in a biologically active form. It isanother object of the present invention to provide host-vector systemsfor the production of a desired protein in native form.

These and other objects and advantages of the present invention, as wellas additional inventive features, will be apparent from the descriptionof the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides an expression vector, the nucleic acidsequence of which comprises a promoter that is capable of controlling,when the vector is in a bacterial host and the temperature is lowered tobelow about 20° C., the production of recombinant peptide or proteinencoded by a gene contained within the vector.

The invention also relates to transformed host cells containing thevectors, particularly transformed E. coli host cells.

In addition, the invention relates to a method of producing in nativeactive form a desired protein encoded by a gene contained within thevector.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the DNA sequence of the pL promoter region including thenucleotides flanking the Eco RI and Bam HI sites (SEQ ID NO:12). Thissequence is also set forth in SEQ ID NO:1.

FIG. 2 depicts the restriction map of plasmid pHG87, which contains thewild-type pL promoter.

FIG. 3 depicts the restriction map of the plasmid pHG91, which containsthe mutant pL promoter pL-9G-50. The sequence of this promoter is alsoset forth in SEQ ID NO:2.

FIGS. 4A-4B depict (A) the presence of the mutant pL promoter pL-9G-50in the plasmid pHG91K and its insertion into phage λB299 by means of adouble crossover recombination event and (B) the resulting vectorlambdaA01107 (λA01107).

FIG. 5 depicts the restriction map of the kanamycin resistant (Km^(R))plasmid pIK86 which was employed in construction of expression vectorsaccording to the invention.

FIG. 6 depicts the restriction map of the plasmid pIK-EL, which containsthe EL cspA promoter.

FIG. 7 depicts the DNA sequence of the EL cspA promoter, which iscomprised approximately of the nucleotides between the arrows labeled"EL". The sequence of the EL cspA promoter contained in pIK-EL is setforth in SEQ ID NO:3 and 13.

FIG. 8 depicts the restriction map of the plasmid pIK-L, which containsthe L cspA promoter.

FIG. 9 depicts the DNA sequence of the L cspA promoter, which iscomprised approximately of the nucleotides between the arrows labeled"L". The sequence of the L cspA promoter contained in pIK-L is set forthin SEQ ID NO:4 and 14.

FIG. 10 depicts the restriction map of the plasmid pIK-M, which containsthe M cspA promoter.

FIG. 11 depicts the DNA sequence of the M cspA promoter, which iscomprised approximately of the nucleotides between the arrows labeled"M". The sequence of the M cspA promoter contained in pIK-M is set forthin SEQ ID NO:5 and 15.

FIG. 12 depicts the restriction map of the plasmid pIK-S, which containsthe S cspA promoter.

FIG. 13 depicts the DNA sequence of the S cspA promoter, which iscomprised of the nucleotides between the arrows labeled "S". Thesequence of the S cspA promoter contained in pIK-S is set forth in SEQID NO:6 and 16.

FIGS. 14A-14B depict (A) the presence of the cspA promoter in theplasmid pIK86 and its insertion into phage λB299 by means of a doublecrossover recombination event and (B) the resulting vector lambdaPcspA(λPcspA).

FIG. 15 depicts the temperature response of the pL-9G-50 promoter fusedto a lacZ reporter gene and integrated in the bacterial chromosome inthe presence (i.e., hip⁺, ) and absence (i.e., hip⁻, ◯) of the positiveregulator of the pL promoter, host integration factor.

FIGS. 16a-16d depict the temperature response of the pL-9G-50 promoterfused to a lacZ reporter gene and integrated in the bacterial chromosomein the presence (i.e., hip⁺, ) and absence (i.e., hip⁻, ◯) of hostintegration factor. Cultures transferred from 37° C. to about 15°-16°C.: (a) β-gal levels, (b) cell density. Cultures transferred from about15°-16° C. to 37° C.: (c) β-gal levels, (d) cell density.

FIG. 17 depicts the expression of lacZ under the control of various cspApromoters at about 15°-16° C.: (□) endogenous cspA mRNA; () EL-lacZmRNA; (▪) M-lacZ mRNA; (▴) S-lacZ mRNA.

FIG. 18 depicts a partial restriction map of plasmid pHG309 (aderivative of plasmid pHG87) and pL-plac (which responds to the lacrepressor and is enhanced by pL promoter elements).

FIG. 19 depicts the expression of lacZ, in terms of enzyme activitiesand messenger RNA level, under the control of various cspA promoters, at37° and 15° C.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to expression vectors which lead to the high levelexpression of subcloned genes in bacterial host cells at lowtemperatures (i.e., at temperatures less than about 20° C.), and whichallow the production of proteins in their native and active form.

The invention is based on the premise that the rate of protein foldingwill be only slightly affected by lowering the temperature to about15°-20° C., while the rate of transcription and protein synthesis, beingbiochemical reactions, will be greatly slowed down (e.g. 5- to 10-fold).Exploitation of these conditions, as in the context of the presentinvention, allows individual proteins sufficient time to refold in anindependent way, yielding active proteins and preventing the formationof aggregates, without reducing the final yield of the desired protein.

Accordingly, the invention provides vectors which allow for theproduction of native and active eukaryotic protein in a bacterial hostcell.

Fermentation in bacterial host cells containing such vectors is based ona two-stage process. At the first stage there is rapid bacterial growthat high temperatures of about 37°-43° C. during which only a very lowlevel of expression of a subcloned gene contained within the vectortakes place. At the second stage there is an increase of gene expressionof the subcloned gene upon transfer to the low temperature. Followingfermentation, pure, soluble and highly active proteins can be isolated.The increased production of protein that occurs upon transfer of thehost cells to low temperature requires utilizing the specificallydesigned expression systems which are the subject of the presentinvention. These vectors allow for increased gene expression andproduction of protein or peptides at a temperature below about 20° C.

By utilizing a high temperature for bacterial growth, the cell carryingthe desired gene will grow rapidly without expressing the desiredpeptide. Following a change to lower temperature in the fermentationsystem, the expression of the gene encoding the foreign protein will beinitiated. Finely tuning the rate of synthesis of the desired protein atlow temperature yields high levels of soluble and biologically activeproteins. This expression system is of general use and can be used forproteins such as viral proteins, especially those useful for preparingvaccines against viruses, in particular against hepatitis viruses suchas hepatitus A, insulin, cytokines such as interleukins such as IL 1-13,in particular IL-2α, interferons, clotting factor genes such as FactorsI-XIII, in particular VIII, hormones such as follicle stimulatinghormones, any glucocerebrosidase such as β-glucocerebrosidase and othergrowth factors and, in principle, any desired protein or peptide.

The vectors of the present invention comprise a nucleic acid sequencewhich comprises a gene encoding a desired protein or peptide, as well asa promoter which is highly active at low temperature. These vectors,upon introduction to a bacterial host cell, enable the high levelproduction of the desired protein or peptide at low temperatures. Theprotein or peptide is obtained in a native active form, and therefore noadditional manipulation of the protein, such as protein refolding, isrequired.

Accordingly, the present invention provides an expression vectorcomprising a nucleic acid sequence which comprises a gene and a promoterthat is capable of controlling, when the vector is in a bacterial hostand the temperature is lowered to below about 20° C., the production ofa recombinant peptide or protein encoded by the gene contained withinthe vector.

In the context of the present invention, a promoter is a DNA sequencethat directs the binding of RNA polymerase and thereby promotes nascentmRNA synthesis. The DNA sequences of eukaryotic promoters differ fromthose of prokaryotic promoters which means that eukaryotic signals maynot be recognized in prokaryotic systems, and vice versa. Accordingly,the promoters of the present invention allow transcription of a genecontained within the vector and, moreover, allow increased transcriptionat temperatures below about 20° C.

Generally the gene inserted into the vector will be foreign (i.e., agene that is not normally expressed in the particular host in which thevector will be introduced), and, preferably, the gene will be ofeukaryotic origin. The gene may be wholly or partially synthetic. Theprotein or peptide encoded by the gene may comprise a full lengthprotein, a polypeptide, or a chimeric protein.

Also, the vectors of the present invention must be capable ofreplication in a bacterial host (i.e., either autonomously or as part ofthe host genome), and must be capable of directing the production of thepeptide or protein in the bacterial host. Thus, the vectors preferablycomprise appropriate control elements (e.g., promoters and ribosomebinding sites) for commanding the production of the peptide or proteinin the host.

In some cases, the gene contained within the vector and encoding theprotein or peptide will contain the appropriate regulatory elements suchthat these elements need not be present on the vector. In other cases(such as when a eukaryotic protein is being produced in a prokaryotichost), it is preferred that the vectors of the present inventioncomprise these additional elements. It is preferred that all of theproper transcription and translation signal be correctly arranged on thevector, such that the foreign gene will be properly expressed within thehost, and protein will be translated, when the temperature is decreasedto below at 20° C.

Accordingly, preferably the expression vectors of the present inventionfurther comprise an initiation codon (such as an ATG codon) positionedso as to allow translation of the peptide or protein to initiate fromthe initiation codon. Even more preferable is that the initiation codonbe positioned between said promoter and said gene.

Also, preferably the expression vectors of the present invention furthercomprise a ribosome binding site positioned so as to control translationof said peptide or protein, preferably positioned between the promoterand the translation initiation codon.

The presence of these additional control elements on the vectors may benecessitated by the fact that translation in prokaryotes, just liketranscription, also does not proceed well in response to eukaryoticsignals. For instance, initiation of translation in prokaryotes requiresthe presence of an initiation codon, such as the ATG codon. If theforeign DNA does not comprise such a codon, one or more such codons canbe provided in the vectors of the present invention. Preferably theinitiation codon will be placed upstream from the sequences throughwhich translation is to initiate, and downstream of the promoters of thepresent invention which impart increased transcription at lowtemperatures upon subcloned fragments placed under their control.

Similarly, the vectors of the present invention may also comprise aribosome binding site (i.e., a "Shine-Dalgarno sequence"; Shine et al.,Nature (1975) 254:34). It is known that efficient translation inprokaryotes is dependent on such a sequence, which exhibitscomplementarity to the 3' end of the 16S ribosomal RNA (rRNA), andlikely promotes binding of mRNA to ribosomes by duplexing with the rRNA,thus allowing the correct positioning of the ribosome on the mRNA.Accordingly, it is preferable that the ribosome binding site becontained within the vectors of the present invention upstream of thetranslation initiation codon and downstream of the promoters whichimpart increased transcription at low temperatures upon subclonedfragments.

Preferably, the vectors of the present invention further compriseadditional operably-bound DNA sequences which contain other controlelements. For instance repressor sites involved in the turning off ofgene expression and enhancers involved in the augmentation of geneexpression are contemplated in the context of the present invention.

The vectors of the present invention comprise a nucleic acid sequencepreferably comprising DNA, which may be single or double-stranded. Thevectors may exist as plasmids, cosmids (i.e., plasmids comprised of acos site) or bacteriophages. In preferred embodiments of the presentinvention, the vector comprises a plasmid, or a bacteriophage, or maycomprise a plasmid:bacteriophage cointegrate which may further beinserted into the bacterial chromosome.

Preferred vectors of the present invention wherein the vector comprisesa bacteriophage are: λPcspA (λA01131 deposited as ATCC No. 75718) andλA01107 (deposited as ATCC No. 75717). Other preferred vectors includebut are not limited to pHG87 and pHG91.

While prokaryotic species such as the bacterium E. coli stop growing atabout 10° C., the vectors of the present invention allow production at atemperature of less than about 20° C. (i.e., at about 15°-16° C.) of ahigh level of a peptide or protein encoded by a gene contained withinthe vector, for example, of the reporter gene lacZ.

The low temperature provides a way to regulate the level of geneexpression, allowing the skilled artisan to find a temperature at whichthe expression rate will be reduced, and providing the maximal timeneeded for proper protein folding, thus preventing protein aggregation.In other words, as the rate of cell growth is slowed down at lowertemperature, the relative time allowed for refolding of individualprotein molecules will be increased.

The expression systems described herein are based on two different setsof promoters and regulatory elements. In one embodiment, the expressionvectors according to the invention comprise the cold-sensitive pLpromoter of phage λ. Descriptions in the literature of the use of the pLpromoter of phage λ and its thermosensitive cI repressor mutant havebeen extensive. Typically, cells are first grown at 30° C. until a highcell mass is reached. At that stage, the cells are transferred to 42° C.and repression of the pL promoter is lifted, leading to the enhancedexpression of a subcloned gene placed under the control of the pLpromoter (see, e.g., Giladi et al., J. Mol. Biol. (1992) 227:985-990;Giladi et al., J. Mol. Biol. (1992) 224:937-948; Ptashne, GeneticSwitch: Phage Lambda and Higher Organisms (Cell Press & BlackwellScientific Publications, Cambridge, Mass. (1992)).

However, the present invention comprises a pL mutant promoter that ismost active at a low temperature (i.e., a temperature less than about20° C.). In addition it was discovered in the context of the presentinvention that the wild-type and improved mutants of the pL promoter(e.g., pL-9G-50) are also highly active at temperatures less than about20° C.

A number of pL derivatives were constructed using conventionalrecombinant DNA techniques and tested for promoter response to lowtemperature as described in Example 2 below. A 3 base pair mutant at theregion from -4 to +4 of the transcription start site (which altered thesequence from GCACATCA to GGACAAGA) diminished the cold-response. Othernucleotide substitutions at other sites within this region, inparticular the sites appearing in bold text, should provide similarresults. A similar reduction in the cold-response was obtained whensequence at the -10 region of the pL promoter was changed from GATACT toTATAAT. Other nucleotide substitutions at other sites within thisregion, in particular the sites appearing in bold text, should providesimilar results. A -9G mutation (in which the sequence GATACT waschanged to GATGCT) resulted in a significant response upon transfer tolow temperature. Other nucleotide substitutions at other sites withinthis region, in particular the site appearing in bold text, shouldprovide similar results.

pHG309, a pL promoter derivative in which an internal deletion, spanningnucleotides -40 to -70, was introduced, retains the response to lowtemperature but does not require IHF for full activity. To obtain the-40 to -78 deletion, two successive PCR reactions were used. In thefirst reaction, a pL fragment extending from -130 to -79 and carrying atthe 3' end an additional 12 base pairs from -39 to -28, was engineeredusng primers #1815 (5'-TCAGAATTCTCACCTACC-3' SEQ ID NO:9) and #2319(5'-TATGTCAACACCTTTTTTATATG-3') (SEQ ID NO:10). In the second reaction,primers #2011 (5'-AAGGATCCAATGCTTCGTTT-3' SEQ ID NO:11) and the productof the first PCR reaction were used. This promoter was found, whenpresent on a multicopy plasmid, to be about 7 to 15 fold more activethan the -9G-50 mutant, depending on growth conditions (as assayed byβ-galactosidase activity). It is expected that other mutants whichcontain lesser deletions in this same area (so long as the open readingframe is maintained) will similarly retain low temperatureresponsiveness and may also may not require IHF for full activity.

The pL-Plac chimeric promoter was generated in a similar way, usingprimer #1815 and a chimeric primer #4402(5'-ACGAGCCGGAAGCATAAAGTGTAAATGTTTTTTATATGAA-3' SEQ ID NO:17) on a pLtemplate in the first PCR reaction. In the second reaction, the productof the first reaction was used as the ustream primer. The universal M13primer was employed and the Plac promoter served as template. In thisfusion product, expression is regulated by the lacI repressor and can beinduced by IPTG. The region from -130 to -79 of pL was fused to Plac(from -36 to +84). The response of this promoter to low temperature wasnot tested.

In another embodiment, the expression vectors of the invention comprisethe cspA promoter. This promoter is highly responsive to lowtemperatures, and, in its native position in the bacterial chromosome,directs the synthesis of over 13% of the bacterial proteins synthesized(Tanabe et al., J. Bacteriol. (1992) 174:3867; Goldstein, Proc. Nat.Acad. Sci. (1990) 87:283). The cspA gene is the major cold shock genethat has been described in bacteria (Goldstein et al., Proc. Natl. Acad.Sci. (1990) 87:283; Jones et al., J. Bacteriol. (1992) 174:3903; Tanabeet al., J. Bacteriol. (1992) 174:3867).

The promoter region of the cspA gene has been dissected, allowing theidentification of a number of regions involved in the enhancement oftranscription. The strength of this promoter allows certain clones to bemaintained only at high temperatures. The influence of the cspA promoteron lacZ expression in the context of the present invention was confirmedas illustrated in the Examples.

A spontaneous mutation in the -35 region (a T:A to C:G mutation at -36)greatly increases the partition of expression between high and lowtemperature (and see FIG. 19). It is expected that any nucleotidesubstitution at this position would provide similar results. It isfurther expected that other mutations ±5 of the -35 site will providesimilar results.

Accordingly, preferred vectors of the present invention comprise a pLpromoter or a PcspA promoter. The pL promoter and PcspA promoter mayfurther comprise a mutation or mutations. In the context of the presentinvention, a mutation results in an alteration of the DNA sequence, andmay consist of an addition, deletion or substitution in the basesequence. Some mutations of the PcspA promoter according to the presentinvention are contained within, for example, pIK-L and pIK-M. Similarly,a pL mutation according to the present invention is contained withinpL-9G-50.

Other preferred vectors of the present invention comprise vectors inwhich the promoter comprises the sequence of: SEQ ID NO:1; SEQ ID NO:2;SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; or SEQ ID NO:6.

Other preferred vectors include: λA01107 contained within the cell linedeposited with the ATCC and assigned No. 75717; λPcspA contained withinthe cell line deposited with the ATCC and assigned No. 75718; pIK-Mcontained within the cell line deposited with the ATCC and assigned No.75719; pIK-EL contained within the cell line deposited with the ATCC andassigned No. 76720; and the plasmids pIK-L and pIK-P.

The present invention further relates to a host-vector system for theproduction of a desired protein in native active form which comprises anexpression vector according to the present invention in a suitablebacterial, and preferably E. coli, host cell.

Also, the expression of the pL promoter is positively regulated by HIF,which is a natural component of E. coli host cells. Accordingly, HIF⁺ E.coli strains, which contain the known host integration factor, HIF, arepreferred for use with the vectors of the present invention comprisingthe pL promoter.

Generally, even more preferred E. coli strains are those which containmutations that will increase the level of protein production and/orstability, or will correct the folding of the desired gene products.Such strains are, for example, mutants defective in the lon or hflgenes, which may improve stability by reducing proteolysis.Additionally, preferred strains may comprise mutations in recA or otherrecombination/repair genes, such mutations which are known to beinvolved in recombination, including deletions, of eukaryotic DNAcontained within bacterial hosts.

Accordingly, the present invention provides a bacterium containing thevector in which the promoter comprises the sequence of: SEQ ID NO:1; SEQID NO:2; SEQ. ID NO:3; SEQ ID NO:4; SEQ ID NO:5; or SEQ ID NO:6.

Similarly, the present invention comprises a nucleic acid comprising avector in which the promoter comprises the sequence of: SEQ ID NO:1; SEQID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; or SEQ ID NO:6.

Other preferred host-vector systems for the production of β-gal innative active form comprise the λPcspA or λA01107 expression vectors ina suitable E. coli host cell, particularly strain A7506.

In a preferred host-vector system, the gene encoding the desired proteinor peptide is integrated into a suitable E. coli host cell chromosomethrough use of the phage λ expression vectors. Integration into thechromosome results in a single copy of the gene fusion which isextremely stable, and which allows for performing the fermentationprocedure without the use of antibiotics.

Genes can be stably introduced into the bacterial chromosome using themethod of the present invention. Under these conditions, the pL promotercan be maintained in the cell in the absence of the cI repressor. A lowlevel of expression is found when the cells are grown at elevatedtemperatures, and a high level of expression is found when the cells aretransferred to a low temperature.

In order to further improve the system so as to allow the use ofplasmids and further increase protein production, a mutant repressorthat is active at high temperature but not at low temperature can beincorporated within the E. coli chromosome or subcloned into a plasmidvector. When supplied with this mutant repressor, cell cultures could begrown at 37° C. before being transferred to lower temperature to allowfor the expression of the cloned gene. It is possible to use the systemas plasmids. It is possible that to acquire greater protein stability,the host has to be grown at 42° C. before lowering the temperature. Itis probable that specific growth conditions will have to be developed,depending on the gene to be expressed. Thus, homologous recombination isnot essential.

In contrast to the pL promoter, positive or negative regulators of thecspA promoter have not been identified. Using the appropriate promoterconstructs according to the present invention, it is possible todirectly manipulate promoter activity. Cells carrying proper fusionswill be grown at 37° C. or higher temperatures, and fusions will beinduced by a change in the culture temperature (i.e., lowering thetemperature below about 20° C.).

The present invention further provides a method of producing a desiredprotein encoded by a subcloned gene contained within an expressionvector which comprises introducing the expression vector into a suitablehost cell, preferably a bacterial host cell, maintaining the cell inculture, and isolating the protein produced.

Cell expansion is facilitated by growing cells between 15° to 45° C.Preferably the cell is maintained in culture at a temperature of about37° C. for a suitable length of time to allow an increase in cellgrowth, and is then maintained in culture at a temperature below about20° C. (between 10° and 20° C.) for a suitable length of time to allowproduction of protein. Cells are grown at elevated temperature followedby transfer to low temperature, while sampling at four hour intervals(up to 72 hours) in order to determine the highest activity. Samples arealso evaluated by protein gel electrophoresis.

Isolation of protein can be by any technique known in the art for thepurification of proteins, such as chromatography, centrifugation,differential solubility, isoelectric focusing, etc. However, preferablythe foreign protein will be obtained from the bacterial host cell bysuitable means whereby the protein is recovered in native active form.Cells are harvested and lysed using standard procedures. It is possiblethat specific buffer conditions will be required to prevent in vitroaggregation of protein.

Standard culture media, rich or minimal, can be used in the context ofthe present invention. As mentioned above, with suitable vectors,addition of antibiotics to the culture media can be avoided, which maybe of particular advantage for production of certain proteins.

The following examples further illustrate the present invention but, ofcourse, should not be considered as in any way limiting its scope.

EXAMPLE 1 Materials and Methods Employed in Experiments

Bacterial strains, phages and plasmids

In the present experiments, standard molecular and genetic techniquessuch as generation of strains, phages and plasmids, gel electrophoresis,DNA manipulations including cloning and sequencing, primer extensionassays, etc., were performed such as are known to those skilled in theart and are described in detail in standard laboratory manuals (e.g.,Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (ColdSpring Harbor, N.Y., 1992); Ausubel et al., Current Protocols inMolecular Biology, (1987); Miller, A Short Course in Bacterial Genetics,(Cold Spring Harbor, N.Y., 1992)). Restriction enzymes and other enzymesused for molecular manipulations were purchased from commercial sources,and used according to recommendations. Strains were cultured, and phageswere maintained, propagated and titered using standard reagents, mediaand techniques (Maniatis et al., Molecular Cloning: A Laboratory Manual,2nd ed. (Cold Spring Harbor, N.Y., 1992); Miller, Experiments inMolecular Genetics (Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1972)). Preferably, Luria-Bertani (LB) Medium containingBacto tryptone (10 g/l), Bacto yeast extract (5 g/l) and NaCl (10 g/l)was employed for growth of cells.

E. coli strain A7506 is a derivative of CSH50 (ara Δ(lac-pro) strA thi;described in Miller, Experiments in Molecular Genetics (Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1972)) which carriesthe λA01107 prophage. Strains A7507, a hip3::cat derivative of A7506,and A7533, a himA82:Tn10 derivative of A7507, were generated by Plvirtransduction.

Strain A6826 is a CSH50 derivative. Strain CSH50 was found to growslowly on minimal medium plates containing the supplements vitamin B₁and proline. A fast growing colony of CSH50 was isolated, purified anddesignated A6826. Strain 7833 is a λcspA EL-lacZ derivative of A6826 andstrain 7832 is a λcspA S-lacZ derivative of A6826.

The DNA sequence of the pL promoter region including the nucleotidesflanking the Eco RI and Bam HI sites is shown in FIG. 1, and is setforth in SEQ ID NO:1. Plasmid pHG87 contains the fragment comprising thewild-type pL promoter, as shown in FIG. 2.

The restriction map of plasmid pHG91 is shown in FIG. 3. Plasmid pHG91contains the mutant pL promoter pL-9G-50. The sequence of this mutantpromoter is characterized by an (A:T) to (G:C) mutation at basepair 220,and a GGCC sequence substituting AATT at basepairs 177-180, and is setforth in SEQ ID NO:2.

Plasmid pHG91 was obtained by fusing the pL-9G-50 promoter to the lacZreporter gene present in plasmid pHG86 (Giladi et al., J. Mol. Biol.(1992) 224:937-948). A kanamycin resistance gene (Km^(R)) was theninserted in the Pst I restriction site located within the bla gene ofpHG91 to generate plasmid pHG91K. When in full length form, the lacZgene encodes β-galactosidase (β-gal) and the bla gene (sometimesdesignated amp) encodes a β-lactamase and confers host cell resistanceto ampicillin.

Phage λA01107 is the product of recombination between phage λB299 andplasmid pHG91K, as set forth in FIG. 4. Phage λB299 (obtained from R.Weisenberg, Section on Microbial Genetics, Laboratory of MolecularGenetics, National Institutes of Health) carries the supF gene flankedby the truncated lacZ and bla genes. Recombination between phage λB299and plasmid pHG91K results in the replacement of the supF gene by thepL-lacZ fusion from pHG91K in phage λA01107.

Plasmids phip⁺, pR87G and pA90D are pEMBL18 derivatives, carrying thewild-type hip gene and its mutant alleles βR87G and βA90D (Menegeritskyet al., J. Mol. Biol. (1993) 231:646-657).

The cspA promoter fragment (309 bp in length from positions -214 to +95;Goldstein et al., PNAS (1990) 87:283-287) was cloned using thepolymerase chain reaction (PCR; PCR Protocols, A Guide to Methods andApplications, Innis et al., eds., Academic Press, Inc. (1990)), and wasinserted into the plasmid pHG86 upstream of the reporter lacZ gene.Plasmid pIK86 (shown in FIG. 5) was constructed from pHG86 by insertingKm^(R) within the bla gene. A set of constructs containing differentsized portions of the cspA promoter region was then prepared from pIK86.The different size fragments carrying the pcspA promoter region wereeach generated by PCR using a lambda phage carrying the cspA chromosomalregion. Two primers were also employed: a primer homologous to the 5'region and carrying an Eco RI restriction site; and a primer homologousto the 3' region and carrying a Bam HI restriction site. The restrictionmaps of the cspA promoter constructs generated were determined.

Of the cspA promoter region-containing plasmids, pIK-EL contains thelargest upstream cspA promoter region, as indicated in FIG. 6. Thesequence of the cspA fragment subcloned in the pIK-EL vector is given inFIG. 7 and SEQ ID NO:3. A smaller cspA promoter fragment was subclonedinto pIK-L, as indicated in FIG. 8. The sequence of the cspA fragmentsubcloned in the pIK-L vector is given in FIG. 9 and SEQ ID NO:4.Similarly, pIK-M contains a still smaller cspA fragment, as indicated inFIG. 10. The sequence of the cspA fragment subcloned in the pIK-M vectoris given in FIG. 11 and SEQ ID NO:5. The smallest cspA promoter fragmentwas subcloned into pIK-S, as indicated in FIG. 12. The sequence of thecspA fragment subcloned in the pIK-S vector is given in FIG. 13 and SEQID NO:6.

Since cspA is a strong promoter, in order to reduce the level ofexpression being driven by the scpA promoter at high temperatures, theseconstructs were subsequently transferred to phage λ by homologousrecombination, as set forth in FIG. 14. The phage was then integratedinto an E. coli chromosome. All crosses between plasmids and λB299 werecarried out in strain A6826.

Preparation of DNA Fragments

DNA fragments used for the primer extension assay were obtained by PCRusing Taq Polymerase (Promega Corp. Madison, Wis.). To obtain the 275-bppL DNA fragment, primer 1921 5'-AAGAATTCGGGTTTTCTTT-3' (pL positions-228 to -217 as set forth in SEQ ID NO: 7), primer 15265'-AAGAGCGTCACCTTC-3' (pL positions +40 to +26 as set forth in SEQ IDNO: 8) and plasmid pHG244 DNA as a template were employed (Giladi etal., J. Mol. Biol., (1992) 224: 937-948). All DNA fragments wereend-labeled with (γ³² P)ATP (Amersham Corp., Arlington Heights, Ill.)and purified on a 5% polyacrylamide gel.

Enzymatic assays

Assays of β-gal specific activity were carried out according to Miller(Miller, Experiments in Molecular Genetics (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1972)).

EXAMPLE 2 Temperature Response of the pL promoter

To test the influence of temperature on gene expression driven by theattenuated pL-9G-50 promoter, this promoter was fused to the lacZ geneand inserted into the E. coli chromosome at the att site using phage λ,and generating strain A7506.

Cultures of strain A7506 were grown in LB medium at various temperaturesto saturation, and were assayed for β-gal activity. As can be seen fromFIG. 15, the levels of β-gal present in the cell were quite high at lowtemperatures (i.e., less than about 20° C.) and declined precipitouslywith increases in temperature. This temperature effect was not observedwhen cultures of strain A7507 were employed. Strain A7507 is a hip⁻derivative of strain A7506 which does not produce host integrationfactor, a positive regulator of the pL promoter. In strain A7507, levelsof β-gal were low at all temperatures.

These results confirm that gene expression driven by the pL-9G-50promoter is induced by low temperatures.

EXAMPLE 3 Temperature Shift Experiments

To study the response of the attenuated pL-9G-50 promoter to temperaturechanges, temperature shift experiments of the lacZ fusion of thispromoter present in the chromosome of strain A7506 were performed.

For these experiments, cultures of strain A7506 (hip⁺) and A7507 (hip⁻)were grown exponentially at 37° C. or about 15°-16° C., and were thentransferred to the alternate temperature (i.e., a culture grown at 37°C. was transferred to about 15°-16° C., and a culture grown at about15°-16° C. was transferred to 37° C.). Levels of β-gal as well as celldensity were determined at different times after the temperature shift.

When exponentially growing A7506 cells were transferred from 37° C. to15°-16° C., a dramatic increase in β-gal levels was observed asindicated in FIG. 16a. This increase occurred despite the long lag incell growth due to the cold shock, as demonstrated in FIG. 16b, and wasdependent on the presence of the wild-type hip gene, as it was notobserved for the A7507 cells under the same conditions.

In contrast, when exponentially growing A7506 cells were transferredfrom 15°-16° C. to 37° C., a rapid cessation in β-gal specific activitywas observed, as indicated in FIG. 16c. This decrease in β-gal specificactivity correlates with the dilution of the existing enzyme by celldivision (as indicated in FIG. 16d), possibly because transcription frompL was turned off rapidly after transfer to the higher temperature.

These results confirm that the attenuated pL-9G-50 promoter is able todrive a high level of gene expression upon transfer from 37° C. to about15°-16° C.

EXAMPLE 4 Effect of Temperature on Promoter Activity

The pL-9G-50 and PcspA promoters were each fused to the lacZ gene in anoperon fusion, transferred to phage λB299 by homologous recombination,and inserted at the att site of the bacterial chromosome as describedabove. Strain N99 was employed as a control to measure plac activityfrom its authentic operon (Ueshima et al., Mol. Gen. Genet. (1989)215:185-189). These experiments were done to determine whether thetemperature response of the pL promoter is unique to that promoter.

For these experiments, cell cultures were grown to saturation in LBmedium at 37° C. or about 15°-16° C., and were then assayed for β-galactivity. Results are as summarized in Table 1.

                  TABLE 1                                                         ______________________________________                                                      β-gal units                                                Promoter        15-16° C.                                                                       37° C.                                        ______________________________________                                        pL-9G-50        3860      260                                                 plac            5950     7320                                                 PcspA           1430      85                                                  ______________________________________                                    

These results confirm that the PcspA promoters as well as the pL-9G-50promoter are able to drive a high level of gene expression at lowtemperatures (i.e., less than about 20° C.). Expression driven by thesepromoters at about 15°-16° C. approaches that driven by the strongpromoter plac. In contrast, expression of plac is reduced about 10-20%at 15°-16° C. as compared with at 37° C.

These results suggest that the higher enzyme levels observed for the pLpromoter at low temperatures are due to increased pL transcriptionrather than to increased stability of the enzyme, or to more efficientprotein translation. Contrary to the plac promoter, the cspA promoterwas 20-fold more active at 15°-16° C. than at 37° C. It is noteworthythat the pL promoter showed the same temperature activation profile asPcspA. This temperature response is an intrinsic property of the pLpromoter, and individual promoters appear to have an inherent responseto temperature which is embedded within the promoter DNA sequence. Theextent of response of the pL promoter to low temperatures, i.e., 14-foldhigher at 15°-16° C. than at 37° C., is similar to that of the cspApromoter, yet these two promoters do not share DNA sequence similaritiesthat indicate why they are more active at low temperature.

Thus, the pL-9G-50 and PcspA promoters minimally drive gene expressionat temperatures at which bacterial cell growth and replication isoptimized (i.e., at about 37° C.). This validates that the promoters canbe employed to obtain maximal gene expression at temperatures at whichbacterial cell growth and replication are minimized (i.e., less thanabout 20° C.).

EXAMPLE 5 Single Copy Chromosomal Fusion by Recombination between Phageλ and Plasmid

Similar experiments as in Example 4 done with the various pIK-cspAconstructs confirm that these also are strong promoters, and that infact, the EL and L constructs are difficult to maintain in host cells,possibly due to lethality as a consequence of a high level of geneexpression. In order to reduce the level of expression driven by thecspA constructs at high temperatures, the constructs were transferred tophage λB299 by homologous recombination (as indicated in FIG. 14A-B).The resultant phages were then integrated into the bacterial chromosome,resulting in a single copy per cell.

As can be seen from Table 2, gene expression driven by the various cspApromoters as reflected in β-gal levels is temperature-regulated and israther high in the resultant strains even though these strains containonly a single copy of the lacZ gene fusions.

                  TABLE 2                                                         ______________________________________                                                     β-gal Units                                                 Single Copy    about 20° C.                                                                     37° C.                                        ______________________________________                                        λIK-EL  20000     1000                                                 λIK-M   24000     2000                                                 λIK-S    9000      900                                                 λIK-P   11170     2440                                                 ______________________________________                                    

The single copy system is extremely stable and allows for performing thefermentation procedure without the use of antibiotics. Similar resultsto those as shown above for the pL promoter demonstrate that the pLwild-type promoter, which cannot be maintained in the absence ofrepressor, can direct the expression of lacZ in a single copy.

These results confirm that the various cspA promoters (as well as thepL-9G-50 promoter) can effectively drive gene expression at lowtemperatures resulting in a high level of translated protein whenpresent as only a single copy integrated into the bacterial chromosome.

EXAMPLE 6 Influence of PcspA promoter on lacZ Expression at LowTemperatures

To test the PcspA promoter influence on lacZ expression, cells carryingthe EL-lacZ fusion (i.e., strain A7833), the M-lacZ fusion (i.e., strainA7831) and the S-lacZ fusion (i.e., strain A7832) on phage λ as a singlecopy in the bacterial chromosome, were examined.

For these experiments, cultures were grown in LB at 37° C., and weretransferred to about 15°-16° C. at time 0. Samples were taken at 1, 2, 4and 6 hours intervals, and RNA was isolated and assayed by primerextension using labeled primers and reverse transcriptase. Radioactivityof the band corresponding to the transcription start site wasdetermined.

As can be seen in FIG. 17, the endogenous cspA mRNA (i.e., □) is presentat high levels only one hour after transfer to the low temperature. Incontrast, EL-lacZ (i.e., ) and M-lacZ (i.e., ▪) mRNAs are maintained athigh levels for longer time periods. Only basal levels of S-lacZ (i.e.,▴) mRNA were observed at all temperatures.

These results confirm that the EL and M promoters may be used to drivegene expression at low temperatures, and that the increased expressionfrom these promoters is reflected in an increased level of transcribedmRNA.

All of the references cited herein are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon apreferred embodiment, it will be obvious to those of ordinary skill inthe art that variations in the preferred methods may be used, includingvariations due to improvements in the art, and that it is intended thatthe invention be practiced otherwise than as specifically describedherein, to encompass these variations. Accordingly, this inventionincludes all modifications encompassed within the spirit and scope ofthe invention as defined by the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 17                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 343 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       CGGGTTTTCTTTGCCTCACGATCGCCCCCAAAACACATAACCAATTGTATTTATTGAAAA60                ATAAATAGATACAACTCACTAAACATAGCAATTCAGATCTCTCACCTACCAAACAATGCC120               CCCCTGCAAAAAATAAATTCATATAAAAAACATACAGATAACCATCTGCGGTGATAAATT180               ATCTCTGGCGGTGTTGACATAAATACCACTGGCGGTGATACTGAGCACATCAGCAGGACG240               CACTGACCACCATGAAGGTGACGCTCTTAAAAATTAAGCCCTGAAGAAGGGCAGCATTCA300               AAGCAGAAGGCTTTGGGGTGTGTGATACGAAACGAAGCATTGG343                                (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 343 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGGGTTTTCTTTGCCTCACGATCGCCCCCAAAACACATAACCAATTGTATTTATTGAAAA60                ATAAATAGATACAACTCACTAAACATAGCAATTCAGATCTCTCACCTACCAAACAATGCC120               CCCCTGCAAAAAATAAATTCATATAAAAAACATACAGATAACCATCTGCGGTGATAGGCC180               ATCTCTGGCGGTGTTGACATAAATACCACTGGCGGTGATGCTGAGCACATCAGCAGGACG240               CACTGACCACCATGAAGGTGACGCTCTTAAAAATTAAGCCCTGAAGAAGGGCAGCATTCA300               AAGCAGAAGGCTTTGGGGTGTGTGATACGAAACGAAGCATTGG343                                (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 290 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "PROMOTER"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CAGACGCGTGAAGCCTTCAAGTGCCGAACTAAAATTGATGCGTTTGATTCAAGCCAACCC60                GGCATTAAGTAAGCAGTTGATGGAATAGACTTTTATCCACTTTATTGCTGTTTACGGTCC120               TGATGACAGGACCGTTTTCCAACCGATTAATCATAAATATGAAAAATAATTGTTGCATCA180               CCCGCCAATGCGTGGCTTAATGCACATCAACGGTTTGACGTACAGACCATTAAAGCAGTG240               TAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCCCTCGTAGTGCA290                         (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 191 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "PROMOTER"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CTTTATTGCTGTTTACGGTCCTGATGACAGGACCGTTTTCCAACCGATTAATCATAAATA60                TGAAAAATAATTGTTGCATCACCCGCCAATGCGTGGCTTAATGCACATCAACGGTTTGAC120               GTACAGACCATTAAAGCAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCC180               CTCGTAGTGCA191                                                                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 140 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "PROMOTER"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TCATAAATATGAAAAATAATTGTTGCATCACCCGCCAATGCGTGGCTTAATGCACATCAA60                CGGTTTGACGTACAGACCATTAAAGCAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCG120               TTGATACCCCTCGTAGTGCA140                                                       (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 121 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "PROMOTER"                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       TTGTTGCATCACCCGCCAATGCGTGGCTTAATGCACATCAACGGTTTGACGTACAGACCA60                TTAAAGCAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCCCTCGTAGTGC120               A121                                                                          (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       AAGAATTCGGGTTTTCTTT19                                                         (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 15 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       AAGAGCGTCACCTTC15                                                             (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 18 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       TCAGAATTCTCACCTACC18                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      TATGTCAACACCTTTTTTATATG23                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      AAGGATCCAATGCTTCGTTT20                                                        (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 356 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      GGCCTCAGCGCCGGGTTTTCTTTGCCTCACGATCGCCCCCAAAACACATAACCAATTGTA60                TTTATTGAAAAATAAATAGATACAACTCACTAAACATAGCAATTCAGATCTCTCACCTAC120               CAAACAATGCCCCCCTGCAAAAAATAAATTCATATAAAAAACATACAGATGACCATCTGC180               GGTGATAAATTATCTCTGGCGGTGTTGACATAAATACCACTGGCGGTGATACTGAGCACA240               TCAGCAGGACGCACTGACCACCATGAAGGTGACGCTCTTAAAAATTAAGCCCTGAAGAAG300               GGCAGCATTCAAAGCAGAAGGCTTTGGGGTGTGTGATACGAAACGAAGCATTGGCC356                   (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 420 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GACAGGATTAAAAATCGATGATTTCGCCCGGGTTTTGGGCGTATCAGTCGCCATGGTAAA60                GGAATGGGAATCCAGACGCGTGAAGCCTTCAAGTGCCGAACTAAAATTGATGCGTTTGAT120               TCAAGCCAACCCGGCATTAAGTAAGCAGTTGATGGAATAGACTTTTATCCACTTTATTGC180               TGTTTACGGTCCTGATGACAGGACCGTTTTCCAACCGATTAATCATAAATATGAAAAATA240               ATTGTTGCATCACCCGCCAATGCGTGGCTTAATGCACATCAACGGTTTGACGTACAGACC300               ATTAAAGCAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCCCTCGTAGTG360               CACATTCCTTTAACGCTTCAAAATCTGTAAAGCACGCCATATCGCCGAAAGGCACACTTA420               (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 420 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      GACAGGATTAAAAATCGATGATTTCGCCCGGGTTTTGGGCGTATCAGTCGCCATGGTAAA60                GGAATGGGAATCCAGACGCGTGAAGCCTTCAAGTGCCGAACTAAAATTGATGCGTTTGAT120               TCAAGCCAACCCGGCATTAAGTAAGCAGTTGATGGAATAGACTTTTATCCACTTTATTGC180               TGTTTACGGTCCTGATGACAGGACCGTTTTCCAACCGATTAATCATAAATATGAAAAATA240               ATTGTTGCATCACCCGCCAATGCGTGGCTTAATGCACATCAACGGTTTGACGTACAGACC300               ATTAAAGCAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCCCTCGTAGTG360               CACATTCCTTTAACGCTTCAAAATCTGTAAAGCACGCCATATCGCCGAAAGGCACACTTA420               (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 420 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GACAGGATTAAAAATCGATGATTTCGCCCGGGTTTTGGGCGTATCAGTCGCCATGGTAAA60                GGAATGGGAATCCAGACGCGTGAAGCCTTCAAGTGCCGAACTAAAATTGATGCGTTTGAT120               TCAAGCCAACCCGGCATTAAGTAAGCAGTTGATGGAATAGACTTTTATCCACTTTATTGC180               TGTTTACGGTCCTGATGACAGGACCGTTTTCCAACCGATTAATCATAAATATGAAAAATA240               ATTGTTGCATCACCCGCCAATGCGTGGCTTAATGCACATCAACGGTTTGACGTACAGACC300               ATTAAAGCAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCCCTCGTAGTG360               CACATTCCTTTAACGCTTCAAAATCTGTAAAGCACGCCATATCGCCGAAAGGCACACTTA420               (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 420 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      GACAGGATTAAAAATCGATGATTTCGCCCGGGTTTTGGGCGTATCAGTCGCCATGGTAAA60                GGAATGGGAATCCAGACGCGTGAAGCCTTCAAGTGCCGAACTAAAATTGATGCGTTTGAT120               TCAAGCCAACCCGGCATTAAGTAAGCAGTTGATGGAATAGACTTTTATCCACTTTATTGC180               TGTTTACGGTCCTGATGACAGGACCGTTTTCCAACCGATTAATCATAAATATGAAAAATA240               ATTGTTGCATCACCCGCCAATGCGTGGCTTAATGCACATCAACGGTTTGACGTACAGACC300               ATTAAAGCAGTGTAGTAAGGCAAGTCCCTTCAAGAGTTATCGTTGATACCCCTCGTAGTG360               CACATTCCTTTAACGCTTCAAAATCTGTAAAGCACGCCATATCGCCGAAAGGCACACTTA420               (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 40 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: other nucleic acid                                        (A) DESCRIPTION: /desc = "SYNTHETIC DNA"                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      ACGAGCCGGAAGCATAAAGTGTAAATGTTTTTTATATGAA40                                    __________________________________________________________________________

What is claimed is:
 1. An expression vector comprising a gene operablylinked to a DNA promoter having the nucleic acid sequence of SEQ IDNO:2, 3, 4, 5 or
 6. 2. The vector of claim 1 wherein said promotercomprises the sequence of SEQ ID NO:2.
 3. The vector of claim 1 whereinsaid promoter comprises the sequence of SEQ ID NO:3.
 4. The vector ofclaim 1 wherein said promoter comprises the sequence of SEQ ID NO:4. 5.The vector of claim 1 wherein said promoter comprises the sequence ofSEQ ID NO:5.
 6. The vector of claim 1 wherein said promoter comprisesthe sequence of SEQ ID NO:6.
 7. The vector of claim 1 which comprisesλA01107 contained within the cell line of ATCC No.
 75717. 8. The vectorof claim 1 which comprises λPcspA contained within the cell line of ATCCNo.
 75718. 9. The vector of claim 1 which comprises plasmid pIK-Mcontained within the cell line of ATCC No.
 75719. 10. The vector ofclaim 1 which comprises plasmid pIK-EL contained within the cell line ofATCC No.
 76720. 11. An E. coli cell containing the vector of claim 2.12. An E. coli cell containing the vector of claim
 3. 13. An E. colicell containing the vector of claim
 4. 14. An E. coli cell containingthe vector of claim
 5. 15. An E. coli cell containing the vector ofclaim
 6. 16. A nucleic acid comprising the vector of claim
 2. 17. Anucleic acid comprising the vector of claim
 3. 18. A nucleic acidcomprising the vector of claim
 4. 19. A nucleic acid comprising thevector of claim
 5. 20. A nucleic acid comprising the vector of claim 6.21. A vector comprising a pL promoter in which the -4 to +4 region shownin FIG. 1 (SEQ ID NO:1) has been mutated from GCACATCA to GGACAAGA. 22.A vector comprising a pL promoter in which the -10 region shown in FIG.1 (SEQ ID NO:1) has been mutated from GATACT to TATAAT.
 23. A vectorcomprising a pL promoter in which the -40 to -78 region shown in FIG. 1(SEQ ID NO:1) has been deleted.
 24. A vector comprising a pL promoter inwhich the -9 region shown in FIG. 1 (SEQ ID NO:1) has been mutated fromGATACT to GATGCT.
 25. A method of producing a protein, comprising thesteps of:transforming a host with a vector comprising a DNA promoterhaving the nucleic acid sequence of SEQ ID NO:2, 3, 4, 5 or 6 operablylinked to a gene encoding a protein, culturing the host at about 37° C.for a time sufficient to allow an increase in culture density, andculturing the host at about 20° C. for a time sufficient to produce saidprotein.
 26. The method of claim 25, wherein said protein is selectedfrom the group consisting of viral proteins, insulin, interleukins,interferons and growth factors.